Injury of the anterior cruciate ligament (ACL) is one of the most important athletic injuries. Histological studies have shown the existence of mechanoreceptors and free nerve endings in the ACL13,17,18). Furthermore, various authors have suggested that the ACL functions as a sensory organ, not only providing proprioceptive information but also initiating protective and stabilizing muscular reflexes3,7,10-12). Accord- ing to Freeman & Wyke’s classification7), four categories of nerve endings can be found in the cruciate ligaments: type I (Ruffini endings), type II (Pacinian corpuscles), type III (Golgi tendon organlike endings), and type IV (free nerve endings) and each of these mechanoreceptors response to
different kind of stimulation20). Also it has been known that the proprioceptive function of the knee decreased in the aged person, and increased in the well trained athlete3). These re- present reversible changes of the sensory function of mecha- noreceptors. So we supposed that it was worthwhile to iden- tify the change of mechanoreceptors of ACL in fixed knee and injured ACL.
MATERIALS AND METHODS
Eighteen white rabbits of New Zealand that were three to five months old and weighed between 2.0 and 3.0 kilo- grams were divided into three groups as control, fixed knee, and injured ACL group. They were kept in the same cir- cumstances with the same feed and place one week before this experiment began. 50 mg/kg of ketamine hydrochlo- ride was infused into the auricular vein for anesthesia for six rabbits of fixed knee group and ACL injured group each on
572 572
The Change of the Mechanoreceptor of Anterior Cruciate Ligament in Fixed Knee and Injured Anterior Cruciate Ligament in the Rabbit
Seong Rae Cho, M.D., Hyoung Min Kim, M.D., Kee Haeng Lee, M.D., Youn Soo Kim, M.D., Joo Hyung Lee, M.D., and Yoon Jong Jahng, M.D.
Department of Orthopaedic Surgery, Holy Family Hospital, The Catholic University of Korea, Bucheon, Korea
572 572 Address reprint requests to
Kee Haeng Lee, M.D.
Department of Orthopaedic Surgery, Holly Family Hospital, The Catholic University of Korea, 2 Sosa-dong, Wonmi-gu, Bucheon 420-717, Korea
Tel: +82.32-340-2260, Fax: +82.32-340-2671 E-mail: [email protected]
Purpose: To identify the change of mechanoreceptor of anterior cruciate ligament (ACL) in fixed knee and injured ACL.
Materials and Methods: Eighteen rabbits were divided into three groups as control, fixed knee, and injured ACL groups. Both knee of all fixed knee group were fixed with K-wire on the first day. The liga- ments of the injured ACL group were cut at tibial attatchment site on the first day. Two rabbits of each group were sacrificed for the evaluation of mechanoreceptor at the 1st, 3rd, and 5th week. Histologic studies were performed by modified gold-chloride stain for the mechanoreceptors
Results: The types of mechanoreceptor in normal ACL were type I, type II, and type III. There was no difference in the appearance rate of each type. In the fixed knee group, the frequency of appearance decreased significantly at 5th week compared with that of the control group. In the injured ACL group, the frequency of appearance decreased at 1st week and showed no difference at 3rd week in compari- son with 1st week, and zero at 5th week. In morphology the degree of degradation of mechanoreceptor increased with time in both experimental group, and showed more severe in the injured ACL group than in the fixed knee group. Most of all mechanoreceptors were located in the subsynovial region, and degrad- ed with synovial degeneration and synovial inflammation.
Conclusion: Mechanoreceptors were changed with the change of intra-articular condition and ligament itself. Therefore the duration of immobilization shoud be shortened if possible and the torn ACL should be repaired anatomically, if indicated, as soon as possible to preserve the mechanoreceptors.
Key Words: Mechanoreceptor, Anterior cruciate ligament (ACL)
the first day of experiment. They were placed in supine posi- tion. Both of knees were fixed with a 0.062 inch Kirshner’s wire which were inserted from the proximal tibia to the dis- tal femur with 45 degrees flexion in fixed knee group. The ACL’s were exposed through the anteromedial approach and were cut at the tibial attachment site in ACL injured group.
Wound were sutured layer by layer. 10 mg/kg of kanamycin was injected intramuscularly for three days after operation to prevent wound infection. Two rabbits of each group were sacrificed and the knee joints were exposed with the same approach as mentioned above at the 1st, 3rd, and 5th week of operation. Gross findings such as status of ACL and syn- ovial membrane were observed. Then whole ACL including femoral attachment site was harvested and modified gold- chloride stain was performed for the histologic evaluation of the mechanoreceptors19). The staining procedure was as follows: 1) The ACL segment was placed in one part 88%
formic acid to three parts filtered, freshly squeezed lemon juice and placed in the dark for 15 minutes. Hereafter, the tissue was handled with paraffin-coated forceps. 2) The lemon juice was decanted off the tissue and the tissue blotted. A 1% solution aqueous gold chloride was added and the tis- sue returned to the dark for 20 minutes. 3) The gold chlo- ride solution was decanted off and 25% formic acid added.
The tissue was kept in the dark overnight, 15-16 hours. 4) The tissue was washed in water and stored in the refrigera- tor. 5) Frozen sections were cut longitudinally into 8 pieces per segment on a sliding microtome at 40 m and stored
in water. 6) The sections were floated on alcoholic gelatin, mounted on slides and allowed to dry. The slides were im- mersed in absolute alcohol for 2 minutes, two changes of xylene, 3 minutes each, and coverslipped. Serial sections were studied with a light microscope (Olympus AHBS-514) under ×40, ×100, and ×200 magnification. We classi- fied the mechanoreceptor by Freeman & Wyke’s classifica- tion7), and measured the type and frequency of appearance of the mechanoreceptors in every section. A mechanorecep- tor was considered to be present when the positively stained one was observed at the same position on 2-3 adjacent seri- al sections or when the morphology of it was definite and it was connected with axon even on one section. We add up the mechanoreceptors from 32 sections and verified the data with Kruskal-Wallis methods. The level of significance was set at p=0.05.
RESULTS
On gross findings, the ACL’s of control group were white, slick, and glitter with approximately 3 mm in thickness and 7 mm in length. In the fixed knee group, most of knee joints showed swelling without relation of time and had synovial hypertrophy and synovitis aggrevated with time. In the in- jured ACL group, severe degenerative change of the synovi- um and ACL was found from the first week of operation.
On microscopic findings, all types of mechanoreceptors except type IV were found at 1st, 3rd, and 5th week (Fig.
1) and there were no differences in the appearance rate of
Fig. 1.Mechanoreceptors of the normal anterior cruciate ligament (modified gold-chloride stain). (A) Most of all mechanoreceptors are located at subsynovial region (×40). (a) Type 1 mechanoreceptor. (b) Type 2 mechanoreceptor. (B) Various mechanoreceptors (×100). (a) Type 1 mechanoreceptor. (b) Type 2 mechanoreceptor. (c) Type 3 mechanoreceptor. (C) Intrasynovial location of type 1 mechanoreceptor (×100). (D) Type 2 mechanoreceptor and its connection axon (×200).
A B C D
a a
a b
c a
b
each type in the control group (Table 1). At 1st week, there were three type I, three type II, and one type III mechanore- ceptors in the fixed knee group and one type I, one type II, and two type III in the injured ACL group. At 3rd week, we found two type I, one type II, and three type III mechanore-
ceptors in the fixed knee group and two type I, three type II, and zero type III in the injured ACL group. There was no significant difference between the 1st and 3rd week in both experimental groups (Table 1). At 5th week, zero type I, one type II, and four type III mechanoreceptors were found
p<0.05.
Normal knee (n=6) Fixed knee (n=6) Injured ACL (n=6)
1 week 3 week 5 week 1 week 3 week 5 week 1 week 3 week 5 week
Type 1 6 3 3 3 2 0 1 2 0
Type 2 2 2 2 3 1 1 1 3 0
Type 3 2 3 3 1 3 4 2 0 0
Total 10 8 8 7 6 5 4 5 0
Table 1.Numbers of mechanoreceptors of anterior cruciate ligament in normal knee, fixed knee, and injured anterior cruciate ligament
Fig. 2.Changes of the mechanoreceptors in the fixed knee (modified gold-chloride stain). (A, B) Mild degradation of synovial tissue and mechanoreceptor at the 1st week of fixation (A, ×40; B, ×200). (C) Moderate degradation of mechanoreceptor at the 3rd week of fixation (×40). (D) Severe degradation of synovial tissue and mechanoreceptor at the 5th week of fixation (×200).
A B C D
Fig. 3.Change of synovial tissue and mechanoreceptor in torn anterior cruciate ligament (modified gold-chloride stain). (A) Early degra- dation of mechanoreceptor at the 1st week of injury (×100). (B) Deep seated mechanoreceptor at the 1st week of injury (×200). (C) Severe degradation of synovial tissue and mechanoreceptor at the 3rd week of injury (×100). (D) Complete degradation of mechanore- ceptor at the 3rd week of injury (×100).
A B C D
in the fixed knee group and no mechanoreceptors in the in- jured ACL group (Table 1). The frequency of appearance of mechanoreceptors decreased with time in fixed knee group and showed significant difference at 5th week compared with that of the control group (p<0.05). And there was also a significant decrease in the frequency at 5th week in the injured ACL group compared with that of the control group (p<0.05).
Mechanoreceptors were mostly located in superficial layer of the ACL and subsynovial region (Fig. 1A-C), and closer to the femoral insertion (Fig. 1A). In morphology the mecha- noreceptors were degraded with time in the fixed knee group (Fig. 2). The degradation of mechanoreceptors was observed from the 1st week (Fig. 3A, B) and the degree of degrada- tion increased at 3rd week (Fig. 3C, D), and mechanorecep- tors were not seen at 5th week in the injured ACL group.
The degree of degradation showed more severe in the injured ACL group than in the fixed knee group. The mechanore- ceptors, were degraded earlier as they were located in super- ficial region of ACL and degraded at the almost same time with degeneration of synovium surrounding ACL. Howev- er, type IV mechanoreceptors were not found in all groups through the entire this study.
DISCUSSION
Morphologic studies have revealed that four categories of nerve endings can be found in the cruciate ligaments. Accord- ing to the Freeman and Wyke’s classification, these are type I, II, III and IV endings7)and the difference in the structure and variability of receptors of some species of mammals had reported16). The type I, Ruffini afferent fibers are slowly adapt- ing, and have low mechanical thresholds. These receptors are able to signal static joint position, intra-articular pressure, and amplitude and velocity of movement. The type II, Pacini- an corpuscles are rapidly adapting and have low thresholds to mechanical stress. They are inactive in the immobile joint and when the joint is rotated at constant speed but become active at acceleration and deceleration. Accordingly, Pacini- an corpuscles are regarded solely as dynamic mechanorecep- tors. The type III, Golgi tendon organlike endings are slow- ly adapting, have high thresholds, and are completely inac- tive in immobile joints. These receptors measure the ten- sion of the ligaments most efficiently. Type IV, free nerve endings which were not a real mechanoreceptors constitute
a pain-receptor system for the tissues of the joint and the paravascular neural fibers have been thought to be concerned with vasomotor control11,12). In this study, type IV, free nerve endings were not found and all types of mechanoreceptors, type I, II, and III were found and were decreased in frequen- cy of appearance in two experimental groups compared with control group.
The human ACL seems to accommodate most of the re- ceptors near the bone attachments, while few receptors are located in the midparts of the ligament. However, there are disagreements about the prime locations for the receptors.
Kenned, et al.13)and Schutte et al.18)reported that they were mainly located closer to the tibial insertion whereas Schultz et al.17)and Girgis et al.9)reported that they were closer to the femoral insertion. Authors cut the tibial end of ACL and harvested the end of the femoral insertion for the experiment because the tibial end of the ligaments were harder to pre- serve during dissection9,17)and the main trunk of the nerve bundle enter and exits from the femoral end of the ligament.
Since 1984, when Schultz et al.17)firstly reported the exis- tence of mechanoreceptors in the human ACL, many related reports have appeared about the function of these mechanore- ceptors. Barrack et al.3)reported that ACL-deficient knee had impaired proprioception and suggested that stabilizing reflex- es originating in ACL receptors might be altered. Miyatsu et al.15)reported that ACL loading has an excitatory effect on the thigh muscles through a multimotor neurone out- put, and that the posterior articular nerve is one of the affer- ent routes from the mechanoreceptors of the ACL. The ACL- muscle reflex may therefore play a physiological role in main- taining knee kinematics. To summarize the function, vari- ous authors have suggested that the ACL functions as a sen- sory organ, not only provideing proprioceptive information but also initiating protective and stabilizing muscular reflex- es3,7,10-12).
The proprioceptive ability decline as part of normal aging and this was found to occur secondary to the arthritic pro- cess. It was also noted that the relative absence of mechanore- ceptors in arthritic knees compared to disease-free knees3,17). Symptoms such as functional instability, feelings of giving way, and muscular weakness sometimes persisted after the various surgical treatment of injured ACL. Freeman et al.6) proposed that such symptoms might be the result of par- tial deafferentation of the capsule and ligaments in associa-
tion with the injured joint. In contrast, studies on athletic populations indicated that extensive training might improve proprioceptive ability2). Therefore, mechanoreceptors are influenced by the conditions of the knee joint.
In the fixed knee group, knee joint swelling persisted re- gardless of the period of fixation and it was thought that the causes were synovitis developed by immobilization and K- wire irritation. The frequency of appearance of receptors was significantly decreased at 5th week and the degree of degra- dation was progressed with time. In the injured ACL group, there were severe inflammatory change in the synovial tis- sue and marked degradation of mechanoreceptors from the 1st week. And no receptors were found at 5th week. As our results, mechanoreceptors of ACL were decreased in number and degraded in morphology with time when the knee joint was fixed and tension of ACL was removed. It was thought that the normal physiologic environment was destroyed, so proprioceptive function declined and the normal sensory feedback was altered by a deafferentation. Adachi et al.1) reported that a positive correlation between the number of mechanoreceptors and accuracy of the joint position sense, suggesting that proprioceptive function of the ACL is relat- ed to the number of mechanoreceptors. Kennedy et al.13)re- ported that the loss of mechanoreceptors due to injury leads to unstable knee function. Krauspe et al.14) demonstrated that electrophysiological afferent responses from the ACL were activated by movement of the knee.
The location of the receptors near the surface of the liga- ments suggested that such a location allowed greater sensi- tivity to their deformation17). The change of intra-articular condition such as an knee effusion can lead to decline the proprioceptive function. Schutte et al.18)insisted that this was due to the responses of mechanoreceptors located in the capsule or menisci but not in the ACL itself. Because the cruciate ligaments are intraarticular but are extrasynovial, so receptors within the cruciate ligaments might be unaf- fected by intraarticular condition and affected only when they were injured directly. Fahrer et al.5)also reported that the knee instability which might be occurred in the case of severe knee effusion was due to the impairment of quadri- ceps muscle contracture. In this study, however, when the knee had hemarthrosis or effusion caused by injury to the ACL, or was fixed, mechanoreceptors in ACL were degrad- ed or disappeared as well as the destruction of synovial tis-
sue because of the subsynovial location of them. It was sup- posed that the number of mechanoreceptors declined earlier in case of the absence of tension to the ligament and hemar- throsis at the same time as in injured ACL group, which was due to the severe degeneration of both synovial tissue and ACL. These results suggested that the mechanorecep- tors of ACL were progressively degraded due to not only the alteration of tension or direct injury to the ACL but also synovial degeneration surrounding ACL, which led to both morhological and functional loss. It was considered that these process was similar to the effect of age and arthrosis on the knee. Clinically the repair of ACL is usually performed with- in two weeks of injury and it is not suitable for direct repair in three weeks later because of degenerative change of ACL.
Therefore, we decided to carry out an experiment on ACL at 1st, 3rd, and 5th week. The results of this study showed that there was significant decrease in number of mechanore- ceptors in both fixed and injured ACL group at 5th week.
Morphologically, there was gradual degradation of the ACL with time in the fixed knee group and severe degradation of both synovial tissue and ACL at 3rd week in injured ACL group.
Authors could not find any articles that reported about re- generation of mechanoreceptors after suture for ruptured ACL on medline. Reconstructive surgery for the treatment of ACL injury is currently widely performed using tendon autografts such as bone-patellar tendon-bone grafts and hamstring grafts, with the objective of an early return to sports. Denti, et al.4)found mechanoreceptors histologically in human ACL remnants obtained arthroscopically. If the ACL remnants could be preserved during reconstruction, the mechanore- ceptors of the remnants might also be preserved to some extent, which may help maintain proprioception after recon- struction. Georgoulis et al.8)and Adachi et al.1)recommend- ed that remnants of the ruptured ACL as a possible source of re-innervation of the ACL autograft should be preserved during ACL reconstruction as much as possible. However, if ACL remnants play a substantial role in proprioceptive function of the injured knee, it seems likely that a normal intact ACL may be still more important.
CONCLUSION
In conclusion, mechanoreceptors were changed with the change of intra-articular condition and ligament itself. There-
fore, the duration of immobilization shoud be shortened if possible and the torn ACL should be repaired anatomically, if indicated, as soon as possible to preserve the mechanore- ceptors. In addition, inflammation of synovial tissue should be treated enthusiastically.
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2. Barrack RL, Skinner HB, Brunet ME and Cook SD: Joint kinesthesia in the high trained knee. J Sport Med Phys Fitness, 24:
18-20, 1983.
3. Barrack RL, Skinner HB and Buckley SL: Proprioception in the anterior cruciate deficient knee. Am J Sports Med, 17: 1-6, 1989.
4. Denti M, Monteleone M, Berardi A and Panni AS: Anterior cruciate ligament mechanoreceptors: histologic studies on lesions and reconstruction. J Orthop Res, 308: 29-32, 1994.
5. Fahrer H, Rentsch HU, Gerber NJ, Beyeler C, Hess CW and Grunig B:Knee effusion and reflex inhibition of the quadriceps. A bar to effective retraining. J Bone Joint Surg, 70-B: 635-638, 1988.
6. Freeman MA, Dean MR and Hanham IW: The etiology and prevention of functional instability of the foot. J Bone Joint Surg, 47- B: 678-685, 1965.
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An anatomical and histological study in the cat. J Anat, 101: 505- 532, 1967.
8. Georgoulis AD, Moebius U, Pappa S, Papageorgiou CO and Soucacos PN:The presence of proprioceptive mechanore- ceptors in the remnants of the ruptured ACL as a possible source of re-innervation of the ACL autograft. Knee Sur Sports Trauma- tol Arthrosc, 9: 364-368, 2001.
9. Girgis FG, Marshall JL and Monajem ARS: The cruciate liga- ment of the knee joint. Anatomical, functional and experimental
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12. Johansson H, Sjolander P and Sojka P: A sensory role for the anterior cruciate ligament. Clin Orthop, 268: 161-178, 1991.
13. Kennedy JC, Alexander IJ and Hayes KC: Nerve supply of the human knee and its functional importance. Am J Sports Med, 10: 329-335, 1982.
14. Krauspe BR, Schmidt M and Schaible HG: Sensory innerva- tion of the anterior cruciate ligament: an electrophysiological study of the response properties of single identified mechanoreceptors in the cat. J Bone Joint Surg, 74-A: 390-397, 1992.
15. Miyatsu M, Atsuta Y and Watakabe M: The physiology of mechanoreceptors in the anterior cruciate ligament, J Bone Joint Surg, 75-B: 653-657, 1993.
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18. Schutte MJ, Dabezies EJ, Zimny ML and Happel LT: Neu- ral anatomy of the human anterior cruciate ligament. J Bone Joint Surg, 69-A: 243-247, 1987.
19. Zimny ML, Onge MS and Schutte M: A modified gold chlo- ride method for the demonstration of nerve endings in frozen sec- tions. Stain Technol, 60: 305-306, 1985.
20. Zimny ML, Schutte M and Dabezies EJ: Mechanoreceptors in the human anterior cruciate ligament. Anat Rec, 214: 204-209, 1986.
목 적: 손상 받은 전방십자인대와 슬관절을 고정했을 때의 전방십자인대에서의 기계적 수용기의 변화를 알아보고자 하였다.
대상 및 방법: 18마리의 토끼를 각각 6마리씩 대조군, 관절고정군, 전방십자인대손상군으로 나누고 실험첫날, 관절고정군 은 K-강선으로 고정하였고, 십자인대손상군은 인대의 경골부착부위를 절단하였다. 실험 제 1주, 3주, 5주에 각 군에서 2마 리씩 도살한 후 조직학적 관찰을 위해 대퇴골부착부를 포함한 전방십자인대를 절취하였다. 기계적 수용기에 대한 조직학 적 관찰은 수정된 gold-chloride 염색법을 이용하였다.
결 과: 대조군의 정상 전방십자인대의 기계적 수용기는 제 1, 2, 3형으로 각 형간 출현 빈도에 차이가 없었다. 관절고정군 에서는 대조군에 비해 제 5주에 기계적 수용기의 빈도가 유의하게 줄었다. 십자인대손상군에서는 수술 후 제 1주에 기계 적 수용기의 수가 대조군에 비해 감소하여 수술 후 제 3주에는 수술 후 1주와 차이를 보이지 않다가 수술 후 5주에는 기계 적 수용기가 보이지 않았다. 기계적 수용기의 형태학적인면에서 실험군 모두에서 시간이 경과함에 따라 붕괴정도가 심해 졌으며, 관절고정군에 비해 전방십자인대군에서 그 정도가 더 심하였다. 기계적 수용기의 대부분은 표층 및 활액조직의 직하부층에 위치하였으며, 전방십자인대를 감싸고 있는 활액막의 변성 및 염증과 함께 거의 동시에 붕괴되었다.
결 론: 전방십자인대의 기계적 수용기는 관절내 조건에 따라 변화하였다. 따라서 기계적 수용기를 보존하기 위해서는 슬 관절의 고정이 필요한 경우 가능한 최소한의 기간만 고정시키고, 전방십자인대의 손상이 있는 경우에는 적응이 된다면 가 능한 한 조기에 해부학적 수복을 해야 한다.
색인 단어: 기계적 수용기, 전방십자인대
토끼에서 슬관절 고정시 및 전방십자인대 손상시 전방십자인대 기계적 수용기의 변화
조성래ㆍ김형민ㆍ이기행ㆍ김윤수ㆍ이주형ㆍ장윤종
가톨릭대학교 성가병원 정형외과
